A solid-phase radioimmunoassay for human beta2-microglobulin.

A simple and relatively rapid as well as highly reproducible determination of beta2-microglobulin is described for general laboratory use. The method is based on a solid-phase radioimmunoassay paper discs as a solid-phase material. The assayable range is approximately in the range of 2 ng to 20 mug of beta2-microglobulin with a precision of approx. 5 percent. This assay requiring only 0.05 ml of urine can be completed within 20 h. Urine in cadmium-exposed workers and residents in cadmium-polluted districts including patients with itai-itai disease were examined and considerable amounts of beta2-microglobulin were observed in these urines.     

Characterization of human beta2-microglobulin by isoelectric focusing.

b2-Microglobulin (B2M) isolated from the urine of normal subjects and patients with cadaveric renal transplantation, showed 2 homologues by isoelectric focusing, one with a pI 5.3, the other with a pI 5.7. These proteins show identical molecular weights by dextran gel filtration and sodium dodecyl sulfate acrylamide gel electrophoresis. The immunogenic reactivity demonstrates partial identity using antiserum to human B2M from 2 different sources.     

Radioimmunoassay of beta2-microglobulin.

A radioimmunoassay for quantitation of beta2-microglobulin in serum and urine is described. Polyethylene glycol 6000 was used to precipitate the antigen-antibody complex. The assay can be performed within 5 hr. In healthy human subjects (mean age, 30.6 years and 42.0 years for serum and urine determinations, respectively) the mean concentration of beta2-microglobulin in serum was 125 nmol/l (range, 49-190 nmol/l). In men the mean 24-hr urinary excretion was 12.9 nmol (range, 8.6-19.2 nmol), and in women it was 5.5 nmol (range, 2.9-8.7 nmol). The assay range was 0.23-19.40 nmol/l, and the detection limit was 0.042 nmol/l, using a prolonged incubation time. The coefficient of variation based on 20 determinations in serum dilutions with a concentration of beta2-microglobulin of 8.14 and 1.92 nmol/l was 8.1 per cent and 9.0 per cent, respectively.     

Serum beta 2-microglobulin in hepato-biliary diseases

The levels of serum beta 2-m in hepato-biliary disease have been examined. Significant increase has been observed in various benign and malignant diseases of the liver, but not in jaundice due to acute pancreatitis. The age-dependent relationship of beta 2-m is present in controls, weaker in the malignant diseases and absent in the benign diseases. The level of beta 2-m is not influenced by jaundice. Serum beta 2-m levels have no role as a discriminant in liver disease.     

Determination of beta 2-microglobulin in human urine and serum by latex immunoassay

This highly sensitive method for determination of beta 2-microglobulin (beta 2-m) in human urine or serum is based on direct agglutination by beta 2-m of latex particles on which an antibody against beta 2-m is adsorbed. The agglutination is quantified by counting the remaining unagglutinated particles, or by turbidimetry. A novel aspect of this method is the capability to prevent nonspecific agglutination of the antibody-coated particles by diluting them with an albumin solution of well-defined characteristics (pH, freshness, concentration) just before the assay. The assayable concentration range is 1--32 micrograms/L, the detection limit 0.5 micrograms/L. Within-assay CV, based on 10 determinations of beta 2-m in urine and serum at two different dilutions, ranged from 4.6 to 8.7%. Between-assay CV, calculated from 10 determinations of beta 2-m in urine and serum, was 10 and 8.4%, respectively. Analytical recovery of beta 2-m in urine averaged 97% and in serum 104% (n = 10). No component of urine or serum interfered. Coefficients of correlation for beta 2-m in urine or serum as measured by radioimmunoassay and latex immunoassay were 0.97 and 0.93, respectively. Concentrations of beta 2-m in serum and urine from 33 healthy men (ages 20 to 67 years) averaged 1.5 mg/l and 54 micrograms/g of creatine, respectively.     

Turnover studies of human beta 2-microglobulin in the rat: evidence for a beta 2-microglobulin-binding plasma protein

1. 125I-labelled beta 2-microglobulin (beta 2m) was injected into rats and protein or non protein-bound radioactivity was determined in plasma urine and several organs. 2. The observed kinetics differed from those expected according to the bicompartmental model for plasma protein turnover. The difference was attributed to the binding of a part of the tracer to a plasma component. Chromatography of plasma taken after injection of beta 2m showed an additional peak of radioactivity at the 55,000-80,000 daltons position. 3. In animals with ligated renal arteries, disappearance of the tracer from the plasma was markedly prolonged, and little non-protein-bound radioactivity was detectable in plasma, indicating that the kidney was the major site of catabolism of beta 2m. 4. Chromatography of plasma from control rats and rats with ligated renal arteries showed that the kidney was the major site of catabolism for free beta 2m only. 5. In normal rats, the urine was found to contain only non-protein-bound radioactivity.     

Serum beta2-microglobulin in liver disease.

The concentration of beta 2-microglobulin in serum was determined in seventy-one patients with various liver disorders. Elevated values were found in most patients with chronic active or chronic persistent hepatitis and in over 80% of patients with alcohol-induced liver cirrhosis. In contrast, patients with alcohol-induced fatty liver, the serum beta 2-microglobulin concentrations were mostly within the normal range. Significant correlation (P less than 0.001) was noted between the elimination rate of galactose from blood and the serum beta 2-microglobulin concentration in patients with alcoholic liver damage but not in patients with chronic hepatitis. The reasons for the increased S-beta 2-microglobulin concentrations in liver diseases are unknown. Several explanations including a release of beta 2-microglobulin from necrotic liver cells or an increased synthesis of beta 2-microglobulin consequent to inflammation in the liver are possible. Alternatively, raised beta 2-microglobulin levels may reflect the hepatic synthesis during reparative growth.     

Serum beta 2-microglobulin in chronic liver diseases

Serum beta 2-microglobulin was determined in 53 patients with chronic liver diseases. No elevation was shown in fatty liver due to obesity or alcoholism. Serum beta 2-microglobulin was abnormal only in 4% of the patients with chronic hepatitis. Determination of serum beta 2-microglobulin seems not useful for the differential diagnosis between chronic hepatitis and fatty liver due to obesity or alcoholism. Serum beta 2-microglobulin was elevated in 29% of the patients with alcoholic liver cirrhosis, in 41% of those with non-alcoholic liver cirrhosis, and in 75% of those with primary liver carcinoma. The average serum beta 2-microglobulin concentration was significantly higher in non-alcoholic liver cirrhosis than in alcoholic liver cirrhosis. There was a significant correlation between serum beta 2-microglobulin and gamma-globulin concentrations in liver cirrhosis.     

Urinary excretion of beta 2-microglobulin in myeloma patients

The levels of beta 2-microglobulin in urine and serum were determined in 39 patients with myelomatosis. In 25 patients the serum beta 2-microglobulin was elevated, and in seven of the patients with increased serum beta-microglobulin the urinary excretion of the protein was also increased. It was concluded that the increased urine beta-microglobulin indicates a renal tubular disorder.     

Stability of alpha 1-microglobulin, beta 2-microglobulin and retinol binding protein in urine

The stability of alpha 1-microglobulin (alpha 1M), beta 2-microglobulin (beta 2M) and retinol binding protein (RBP) in urine was determined in 135 random samples from children with renal disease, febrile illness, malignancy, and from controls. Immediately after voiding, samples were divided into two portions, one of which was alkalinized. After identical transit times and laboratory handling the pH and concentrations of the individual proteins in each pair were measured. beta 2M was unstable in urine of pH less than 7 and grossly so below pH 6. In some instances beta 2M was low or undetectable even in the alkalinized samples when alpha 1M and RBP levels were raised, suggesting that degradation of beta 2M may have occurred prior to voiding. Concentrations of alpha 1M and RBP were significantly lower in the non-alkalinized fractions at pH less than 7, although to lesser degree than for beta 2M. Contrary to previous reports, we conclude that the stability of all 3 proteins is affected by urinary pH and recommend that this be measured and alkalinisation performed at the time of voiding.     

Catabolism of rat beta2-microglobulin in the rat.

Highly purified rat beta2-microglobulin (beta2m) as well as cytochrome c and lysozyme were radiolabeled and their catabolism studied in the rat. More than 90 percent of these low molecular weight proteins were removed from the serum within an hour and excreted into the urine by 24 hours. Except for the kidney in which the concentration of these protein is ten- to twentyfold greater than in the serum, there is little evidence that rat tissues are concentrating these proteins. The stomach was found to concentrate radioiodine. The catabolism of rat beta2m differed from that of cytochrome c and lysozyme in that the kidney contained twice as much labeled rat beta2m. In addition, the rat excretes 10 to 15 percent of the injected rat beta2m but only 1 to 5 percent of the cytochrome c or lysozyme. These studies established a basis for turnover studies of beta2m complexed with other cell membrane proteins, for example, HL-A or H-2 peptides.     

Serum beta2-microglobulin in controls and cancer patients.

Serum beta2-microglobulin levels have been measured in 210 cancer and control patients to assess the significance of this investigation in cancer patients. Subjects studied included patients with breast and gastrointestinal cancer, corresponding control patients in both categories, and healthy volunteers. The composition of these groups allowed an assessment of the relative importance of changes related to cancer, benign disease, age and sex. A significant rise in serum beta2-microglobulin levels with advancing age was demonstrated in the control subjects. Mean levels were also consistently higher in females than in males in each patient group. After statistical correction for these age and sex effects, mean values remained significantly higher in each of the various cancer groups than in their controls. Patients with more advanced breast cancer had higher levels than those with 'early' disease, as did patients with stomach cancer compared to those with colo-rectal cancer. One possible interpretation is that levels increase with increasing tumour bulk, and therefore the estimation of serum beta2-microglobulin may be useful as one of a battery of tests in the management of cancer patients.     

Selective elution of HLA antigens and beta 2-microglobulin from human platelets by chloroquine diphosphate

To determine whether chloroquine can specifically elute HLA antigens and beta 2-microglobulin (beta 2-M) from the platelet surface, quantitative immunofluorescence flow cytometry and monoclonal antibodies were used to show that HLA antigens and beta 2-M were proportionally eluted from the platelet surface without affecting the membrane glycoproteins IIb and IIIa. Second, an autoradiogram of electrophoresed I125-labeled platelets showed that only beta 2-M but not other I125-labeled membrane proteins could be eluted. Although HLA antigens were poorly labeled by I125 and could not be detected on the autoradiogram, the eluted HLA antigens could be detected by anti-HLA monoclonal antibody and immunoblotting techniques. No loss of plasma membrane integrity was observed by transmission electron microscopy after chloroquine treatment of platelets. The results indicate that chloroquine selectively elutes HLA antigens and their noncovalently associated beta 2-M without affecting other integral platelet membrane proteins.     

Double monoclonal time-resolved immunofluorometric assay of beta 2-microglobulin in serum

Previously reported immunochemical assays of beta 2-microglobulin (beta 2m) have usually been based on polyclonal antisera. We have developed a "sandwich"-type time-resolved immunofluorometric assay (TR-IFMA) for beta 2m in serum, based on two monoclonal antibodies against human beta 2m. Microtiter wells are coated with the capture antibody, and the tracer antibody is labeled with a europium chelate. In a simple and fast assay procedure, prediluted serum samples are incubated with the tracer for 1 h in the microtiter wells, after which the wells are washed and the fluorescence of Eu is measured. The mean analytical recovery was 101.8% and results by TR-IFMA showed a good linear correlation with those by an established radioimmunoassay. The analytical range of TR-IFMA is large and well suited for clinical purposes.     

Circulating levels of beta 2-microglobulin in the over 70s

The median serum beta 2-microglobulin value for a population aged over 70 years was 2.30 mg/l. This value increased significantly with age in both men and women--there were no sex differences.